The nonapeptide bradykinin (BK) and the homologous decapeptide kallidin (Lys-BK) are produced endogenously by enzymatic cleavage by plasma and tissue kallikreins of their circulating precursor proteins (kininogens) in many tissues and under a wide variety of conditions for regulation of both normal and abnormal physiology. Bhoola et al., 44 Pharmacol Revs. 1 (1992). In addition to their functions in regulation of normal physiology, there is considerable evidence to support the hypothesis that these peptides (collectively called kinins) are the initiators of most, if not all, inflammation. Stewart, 42S Agents Actions 145 (1993). Trauma, infection and allergic reactions have all been shown to stimulate kinin release; the kinins then stimulate release of the further chain of inflammatory mediators, such as prostaglandins, TNF and various interleukins. A decade of animal studies and recent clinical trials have indicated that BK antagonists may become important drugs for anti-inflammatory medicine.
Under normal conditions, actions of kinins are transitory in vivo, due to rapid cleavage of the peptides by several enzymes. The most important of these are angiotensin converting enzyme (ACE; Kininase II), localized principally to the endothelium of the pulmonary vasculature, and the soluble circulating carboxypeptidase N (CPN; kininase I). The membrane-bound enkephalinase (endopeptidase 3.4.24.11) and aminopeptidase P (APP) are less active toward the kinins. ACE removes the C-terminal dipeptide from BK, yielding BK-(1-7), which is inactive biologically. The products of CPN action, BK-(1-8) and kallidin-(1-9), while inactive at B2 receptors, are the normal ligands for B1 receptors. Products of cleavage by APP and endopeptidase 24.11 are totally inactive. ACE normally cleaves more than 99% of BK on a single passage through the pulmonary circulation, and CPN normally causes BK to have a plasma half-life of 15 seconds or less.
Biological actions of kinins are mediated by two classes of receptors: B1 and B2. Both classes of receptors have been cloned and sequenced from a variety of species. They are typical G protein-coupled receptors having seven putative helical membrane-spanning segments. In various tissues, BK receptors are coupled to every known second messenger system. Prominent among these, and particularly important in inflammation, are phospholipase A2 (PLA2), with subsequent production of prostaglandins and leukotrienes, and phospholipase C (PLC), with subsequent stimulation of cell proliferation, for wound healing. B2 receptors are constitutively expressed on the membranes of most cells, and require the full chain of the kinin peptides, including the C-terminal arginine residue, for binding and activation. In contrast, B1 receptors are not normally expressed in most tissues; their expression is stimulated in inflammation. Marceau, 30 Immunopharmacol. 1 (1995). Activation by kinins of vascular B1 and B2 receptors causes vasodilation and lowering of blood pressure. The severe fall in blood pressure (shock) of systemic bacterial infection appears to be initiated and sustained by production of BK. Bacterial enzymes produce BK, either by direct cleavage of circulating kininogens or by activation of kallikreins which then cleave kininogens. Sakada, et al., 33 Immunopharmacol 377 (1996). The lipopolysaccharide (LPS) endotoxin of Gram-negative bacterial cell walls also stimulates production of BK and initiates release of TNF and lymphokines. A particularly vicious aspect of infection is that ACE is lost from the pulmonary circulation, causing kinins to be metabolized principally by CPN, thus producing large amounts of the ligands for the concomitantly induced B1 receptors and causing shock.
Antagonists for BK B2 receptors were introduced in 1984 (Vavrek and Stewart, 6 Peptides 161 (1985) and stimulated a renaissance of kinin research. Rapid metabolism of kinins had made demonstration of physiological and pathophysiological roles for kinins very difficult. With tools available to block kinin receptors, demonstrations of participation of kinins in regulation of every major physiological system and initiation or mediation of much pathophysiology soon followed. The essential structural change in the BK molecule for production of antagonists was replacement of the 7-proline residue by a D-aromatic amino acid, most commonly D-Phe. This change yielded a weak partial antagonist. Additional changes to increase receptor affinity and decrease enzyme degradation yielded the useful "first generation" B2 antagonists (NPC-349; see Table 1). These antagonists had low affinity for BK receptors and showed short activity in vivo, due to cleavage by CPN. Stewart and Vavrek in "Bradykinin Antagonists,: Burch RM, Ed., Pergamon, Oxford 51 (1990). Regoli et al., 123 Eur. J. Pharmacol 61 (1986).
Although B1 antagonists had been described earlier (Regoli et al., 55 Can J. Physiol Pharmacol 855 (1977), they did not attract much interest until the demonstration that B1 receptors, normally not present in most tissues, are expressed in chronic inflammation. Perkins et al., 53 Pain 191 (1993); Marceau, 30 Immunopharmacol. 1 (1995). Replacement of the C-terminal phenylalanine in the normal B1 ligands BK-(1-8) and kallidin-(1-9)! by a hydrophobic aliphatic amino acid yielded the first B1 antagonists. An example is Leu8!-BK(1-8). The "first generation" B1 antagonists, like the earliest B2 antagonists, were rapidly degraded in vivo.
The "second generation" of B2 antagonists was begun with introduction by Hoechst investigators of Icatibant (HOE-140) (Hock et al., 102 Brit J Pharmacol 769 (1991) and followed by the Cortech Bradycor (CP-0127) (Cheronis et al., 35 J Med Chem 1563 (1992) (see Table 1). In the first generation B2 antagonists, such as the Stewart NPC-349, although the D-amino acid residue at position seven blocked action of ACE, and the N-terminal D-arginine residue blocked aminopeptidase action, these antagonists were still degraded by plasma CPN and by endopeptidase 24.11. Indeed, the first generation B2 antagonists, such as NPC-349, showed B1 antagonist activity in vivo, due to enzymatic removal of the C-terminal arginine (Regoli et al., 123 Eur. J. Pharmacol 61 (1986). The significant structural feature of Icatibant is the incorporation of imino acids, which greatly restrict peptide conformation and inhibit enzyme action, at positions seven and eight. Incorporation of octahydroindolecarboxylic acid (Oic) at position eight made this peptide resistant to cleavage by CPN and thus greatly extended its in vivo activity. The bulky D-tetrahydroisoquinolinecarboxylic acid (Tic) at position seven, combined with Oic8, strongly restricts the conformational freedom of the important carboxyl end of the peptide to a shape evidently preferred by the B2 receptors. Kyle et al., 36 J Med Chem 1450 (1993). The very hydrophobic nature of these residues is probably also important, causing Icatibant to have a slow "on-time" and a very long persistence at or near receptors. Bradycor owes its increased potency to its dimeric nature, with perhaps some additional contribution from the hydrophobic character of the linker moiety. Despite these improvements, both of these antagonists are slowly degraded by plasma and tissue extracts. Endopeptidase 24.11 is probably important in this degradation.
Recently a dramatic improvement (the "third generation" of BK antagonists) came with introduction of .alpha.-(2-indanyl)glycine (Igl) into the antagonist structure. Stewart et al., 33 Immunopharmacol 51 (1996). An extremely interesting peptide is B9430, which has L-Igl at position five, D-Igl at position seven, and Oic at position eight. This antagonist shows truly impressive high potency and long duration of action in vivo. The Igl residue at position five evidently blocks degradation by endopeptidase 24.11. These new antagonists persist more than six hours in plasma and tissue homogenates, and show very long duration of action in vivo. A single intravenous injection of B9430 in rats blocks the hypotensive action of BK for more than four hours, a subcutaneous injection in rats can block BK action for 48 hours, and a subcutaneous injection in rabbits blocks BK action for more than 24 hrs. Perhaps the most remarkable property of the antagonists containing Igl is their high potency at B1 receptors, in addition to the anticipated B2 activity, although they contain the C-terminal arginine residue that normally prevents B1 receptor activity of agonists and antagonists. Activity of these new antagonists at both receptors has been demonstrated in cultured cells, in isolated smooth muscle tissues, and in vivo. They are active at human B1 and B2 receptors. Most recently, B9430 has been shown to be active following intragastric administration in rats, although the bioavailability is low. Whalley et al., 75 Can J Physiol Pharmacol 629 (1997). This result suggests that we may have made progress on the way toward the ambitious goal of a chemically modified peptide having significant oral activity.
Bradykinin has important growth factor activity, although the lability of BK has made demonstration of this property difficult. Production of BK in trauma (BK is produced whenever blood clotting is initiated) is probably to stimulate wound repair, where it can act in concert with platelet-derived growth factor. Recent papers have begun to delineate the intracellular events, especially tyrosine phosphorylation, that follow action of BK on cells. Tallett et al., 17 Peptides 665 (1996). Small cell lung cancer (SCLC) cells express BK receptors, and evidently use BK and other peptides (substance P, bombesin) as growth stimulants. Woll and Rozengurt, 85 Proc Nat Acad Sci US 1859 (1988). Several peptide antagonists have been tested as potential inhibitors of SCLC growth, and some progress has been reported. Staley et al., 12 Peptides 145 (1991). Our BK antagonists have been tested consistently by Dan Chan at the University of Colorado Cancer Center for their effects on cultured cells of SCLC. While all our good antagonists, especially the new "third generation" peptides, block the BK-evoked increase in intracellular calcium concentration (Bunn et al., 54 Cancer Res 3602 (1994), they do not inhibit cell growth. Most recently, however, dimers of our new antagonists, such as B9870, were found to inhibit growth of cultured SCLC cells. Chan et al., 33 Immunopharmacol 201 (1996).